The aim of the proposed project is to study the structure and function of the DNA-protein covalent complex in Bacillus subtilis bacteriophage phi 29. We will study: a) The way by which sus3 mutants initiate DNA replication by labeling sus3-infected B. subtilis su- with 35S-sulfate and 3H-thymidine, isolation of the protein-DNA complex and analysis of the protein by polyacrylamide gel electrophoresis in the presence of SDS. b) Search for a precursor of protein p3. Pulse-chase experiments in B. subtilis infected minicells in the presence of amino acid analogs or of protease inhibitors will be carried out. c) Purification of protein p3, either from phage particles to be used for structural studies and to prepare monoclonal antibodies or from phage-infected cells to be obtained in a native state for further in vitro studies. d) Mechanism of initiation of phi 29 DNA replication; possible role of protein p3 as a primer. The in vivo formation of a covalent linkage between protein p3 (or precursor) and dAMP will be studied by adding 3H-deoxyadenosine to cells infected with a DNA elongation mutant. The in vitro formation of such a linkage will be also studied using the purified native protein p3 and 3H-dATP in the presence or absence of protein p3-containing phi 29 DNA and extracts from infected or uninfected cells. e) Amino acid sequence of protein p3. It will be derived from the DNA sequence of cistron 3, once the amino acids corresponding to the amino- and carboxy-termini are determined in protein p3.